We designated these sets as 29 M-miRs and 17 Pr-miRs. To validate Pr-miR and M-miR, we applied them to patients in the alternative dataset. This analysis was performed using the unsupervised first component of a principal component analysis. At different cutoff points of the unbiased Pr-miRs and M-miR-derived classifiers, the combinations of sensitivities and specificities reflect their ability to discriminate between the oligo- vs polymetastatic tissue samples thus are plotted as receiver operating characteristic curves in Fig. 2. Next, we investigated whether specific microRNAs differentially expressed between oligometastatic and polymetastatic patients were associated with phenotypic Cycloheximide Small Molecules inhibitor change from oligo- to polymetastases. Since metastatic development is a multi-step process and all patients by definition had metastasis at time of radiation treatment, we hypothesized that late events in the metastatic process were likely to account for differences in the oligo- and polymetastastic phenotypes. Primary tumors are likely more heterogeneous with respect to cells with metastatic potential, thus we focused on the prioritized microRNAs derived from the metastatic tissue samples. We rank ordered the 29 prioritized microRNAs obtained from metastatic tissue according to fold change. As shown in Table 1b, the two microRNAs with highest fold changes, miR-654-3p and miR-654-5p, are produced in the cells by two-complementary/opposite strands of the same precursor microRNAs. Their joint expression suggests a common transcriptional event likely unrelated to their specific function. MicroRNA-200c has anti- or pro-metastatic functions depending on at which point in the metastatic cascade it acts. For example, it inhibits the invasiveness of cancer cells at the primary site by suppressing epithelial to mesenchymal transition, while it LY2109761 abmole enhances colonization efficiency at distant metastatic sites by promoting the reversion from EMT to mesenchymal-to-epithelial-transition. To demonstrate prioritized microRNAs from the clinical samples are functionally important, as a proof of principle, we examined whether microRNA-200c may regulate oligo- to polymetastatic progression. We specifically enhanced the function of this microRNA via synthetic mimics in the most stable oligo-like L1-R2 cell line prior to tail vein injection. Whereas injection of non-treated or control mimics-treated L1-R2 cells produced predominantly oligometastases or no macroscopic metastasis, increased expression of microRNA-200c in the L1-R2 cell line produced significantly more mice with polymetastases, one-tailed Mann Whitney U, for polymetastases compared to controls. Real-time imaging visualization and histological characterization also confirmed this conversion. Since microRNA-200c has mainly been characterized as a metastasis suppressor, our prediction of its role in promoting oligoto polymetastatic progression is novel.